The HPV E2 Transcriptional Transactivation Protein Stimulates Cellular DNA Polymerase Epsilon

The papillomavirus (PV) protein E2 is one of only two proteins required for viral DNA replication. E2 is the viral transcriptional regulator/activation protein as well as the initiator of viral DNA replication. E2 is known to interact with various cellular DNA replication proteins, including the PV E1 protein, the cellular ssDNA binding complex (RPA), and topoisomerase I. Recently, we observed that cellular DNA polymerase ε (pol ε) interacts with the PV helicase protein, E1. E1 stimulates its activity with a very high degree of specificity, implicating pol ε in PV DNA replication. In this paper, we evaluated whether E2 also shows a functional interaction with pol ε. We found that E2 stimulates the DNA synthesis activity of pol ε, independently of pol ε’ s processivity factors, RFC, PCNA, and RPA, or E1. This appears to be specific for pol ε, as cellular DNA polymerase δ is unaffected by E1. However, unlike other known stimulatory factors of pol ε, E2 does not affect the processivity of pol ε. The domains of E2 were analyzed individually and in combination for their ability to stimulate pol ε. Both the transactivation and hinge domains were found to be important for this stimulation, while the E2 DNA-binding domain was dispensable. These findings support a role for E2 beyond E1 recruitment in viral DNA replication, demonstrate a novel functional interaction in PV DNA replication, and further implicate cellular pol ε in PV DNA replication

The HPV E2 Transcriptional Transactivation Protein Stimulates Cellular DNA Polymerase Epsilon

The papillomavirus (PV) protein E2 is one of only two proteins required for viral DNA replication. E2 is the viral transcriptional regulator/activation protein as well as the initiator of viral DNA replication. E2 is known to interact with various cellular DNA replication proteins, including the PV E1 protein, the cellular ssDNA binding complex (RPA), and topoisomerase I. Recently, we observed that cellular DNA polymerase ε (pol ε) interacts with the PV helicase protein, E1. E1 stimulates its activity with a very high degree of specificity, implicating pol ε in PV DNA replication. In this paper, we evaluated whether E2 also shows a functional interaction with pol ε. We found that E2 stimulates the DNA synthesis activity of pol ε, independently of pol ε’ s processivity factors, RFC, PCNA, and RPA, or E1. This appears to be specific for pol ε, as cellular DNA polymerase δ is unaffected by E1. However, unlike other known stimulatory factors of pol ε, E2 does not affect the processivity of pol ε. The domains of E2 were analyzed individually and in combination for their ability to stimulate pol ε. Both the transactivation and hinge domains were found to be important for this stimulation, while the E2 DNA-binding domain was dispensable. These findings support a role for E2 beyond E1 recruitment in viral DNA replication, demonstrate a novel functional interaction in PV DNA replication, and further implicate cellular pol ε in PV DNA replication

Optimization of 2-Acylaminocycloalkylthiophene Derivatives for Activity against Staphylococcus aureus RnpA

Staphylococcus aureus is well-recognized to cause debilitating bacterial infections that are difficult to treat due to the emergence of antibiotic resistance. As such, there is a need to develop new antimicrobials for the therapeutic intervention of S. aureus disease. To that end, S. aureus RnpA is an essential enzyme that is hypothesized to participate in two required cellular processes, precursor tRNA (ptRNA) maturation and mRNA degradation. Corresponding high throughput screening campaigns have identified the phenylcarbamoyl cyclic thiopenes as a chemical class of RnpA inhibitors that display promising antibacterial effects by reducing RnpA ptRNA and mRNA degradation activities and low human cell toxicity. Herein, we perform a structure activity relationship study of the chemical scaffold. Results revealed that the cycloalkane ring size and trifluoroacetamide moiety are required for antibacterial activity, whereas modifications of the para and/or meta positions of the pharmacophore’s phenyl group allowed tuning of the scaffold’s antimicrobial performance and RnpA inhibitory activity. The top performing compounds with respect to antimicrobial activity also did not exhibit cytotoxicity to human cell lines at concentrations up to 100 µM, greater than 100-fold the minimum inhibitory concentration (MIC). Focused studies of one analog, RNP0012, which exhibited the most potent antimicrobial and inhibition of cellular RnpA activities revealed that the compound reduced bacterial burden in a murine model of S. aureus disease. Taken together, the results presented are expected to provide an early framework for optimization of next-generation of RnpA inhibitor analogues that may represent progenitors of a new class of antimicrobials